Nucleic acid molecules encoding starch phosphorylase from maize

ABSTRACT

Nucleic acid molecules are described which encode enzymes involved in the starch synthesis in plants. These enzymes are starch phosphorylases from maize. The invention further relates to vectors containing such nucleic acid molecules and to host cells transformed with the described nucleic acid molecules, in particular to transformed plant cells and to plants which may be regenerated therefrom and which exhibit an increased or reduced activity of the described proteins.

This application is a continuation of international applicationPCT/EP98/0 1183, filed on Mar. 3, 1998, which designated the UnitedStates.

FIELD OF THE INVENTION

The present invention relates to nucleic acid molecules encoding astarch phosphorylase from maize. Furthermore, the present inventionrelates to vectors, bacteria as well as to plant cells transformed withthe described nucleic acid molecules and to the plants containing thesame. Moreover, methods for the production of transgenic plants aredescribed which, due to the introduction of DNA molecules encoding astarch phosphorylase from maize, synthesize a starch which is modifiedin its properties.

With respect to the increasing significance which has recently beenascribed to vegetal substances as regenerative sources of raw materials,one of the objects of biotechnological research is to try to adaptvegetal raw materials to the demands of the processing industry. Inorder to enable the use of regenerative raw materials in as many areasas possible, it is furthermore important to obtain a large variety ofsubstances. Apart from oils, fats and proteins, polysaccharidesconstitute the essential regenerative raw materials derived from plants.Apart from cellulose, starch maintains an important position among thepolysaccharides, being one of the most significant storage substances inhigher plants. Among those, maize is one of the most interesting plantsas it is the most important cultivated plant for the production ofstarch.

The polysaccharide starch is a polymer made up of chemically homogeneousbasic components, namely the glucose molecules. However, it constitutesa highly complex mixture from various types of molecules which differfrom each other in their degree of polymerization and in the degree ofbranching of the glucose chains. Therefore, starch is not a homogeneousraw material. One differentiates particularly between amylose-starch, abasically non-branched polymer made up of α-1,4-glycosidically branchedglucose molecules, and amylopectin-starch which in turn is a complexmixture of various branched glucose chains. The branching results fromadditional α-1,6-glycosidic interlinkings. In plants used typically forthe production of starch, such as maize or potato, the synthesizedstarch consists of approximately 25% amylose-starch and of about 75%amylopectin-starch.

In order to enable as wide a use of starch as possible, it seems to bedesirable that plants be provided which are capable of synthesizingmodified starch which is particularly suitable for various uses. Onepossibility to provide such plants—apart from breeding methods—is thespecific genetic modification of the starch metabolism ofstarch-producing plants by means of recombinant DNA techniques. However,a prerequisite therefore is to identify and to characterize the enzymesinvolved in the starch synthesis and/or the starch modification as wellas to isolate the respective DNA molecules encoding these enzymes.

The biochemical pathways which lead to the production of starch arebasically known. The starch synthesis in plant cells takes place in theplastids. In photosynthetically active tissues these are thechloroplasts, in photosynthetically inactive, starch-storing tissues theamyloplasts.

The most important enzymes involved in starch synthesis are starchsynthases as well as branching enzymes. In the case of other enzymes andalso, for example, in the case of starch phosphorylases, their preciserole during starch biosynthesis is unknown.

In order to provide further possibilities in order to modifystarch-storing plants in such a way that they synthesize a modifiedstarch, it is necessary to identify DNA sequences encoding furtherenzymes involved in the starch biosynthesis, such as starchphosphorylase. Such proteins are known, for example, from Vicia faber(Buchner et al., Planta 199 (1996), 64-73), Solanum tuberosum (St.Pierre and Brisson, Plant Science 110 (1995), 193-203; Sonnewald et al.,Plant. Mol. Biol. 27 (1995), 567-576; Bhatt and Knowler, J. Exp. Botany41 (Suppl.) (1990), 5-7; Camirand et al., Plant Physiol. 89 (4 Suppl.)(1989), 61), Ipomoea batatas (Lin et al., Plant Physiol. 95 (1991),1250-1253), sugar beet (Li et al., Ohio J. of Sci. 90 (1990), 8),spinache and maize (Mateyka and Schnarrenberger, Plant Physiol. 86(1988), 417-422) as well as pea (Conrads et al., Biochim. Biophys. Acta882 (1986), 452-464).

They are characterized as enzymes catalyzing the reversiblephosphorylysis of terminal glucose units of α-1,4-glucans according tothe following equation:

glucan_(n)+P_(i)|⇄glucose-1-phosphate+glucan_(n−1)

Depending on the relative concentration of P_(i) and glucose-1-phosphate(G1P), the enzyme may have a degrading or, as the case may be,synthesizing effect on the glucans (Waldmann et al., CarbohydrateResearch 157 (1986), C4-C7). On the basis of the differences in thelocalization, in the affinities to the glucans and in the regulation andthe size of monomers, the plant starch phosphorylases are classified asfollows:

Type 1: situated within the cytosol of plant cells; very high affinityto longer-chained branched glucans; unregulated; monomeric size ofapproximately 90 kD;

Type 2: situated within the plastids of plant cells; affinity tomaltodextrines; low affinity to polyglucans; unregulated; monomeric sizeof approximately 105 kD.

DNA sequences encoding the corresponding starch phosphorylases havesofar been isolated only from a small number of plant species such aspotato (Buchner et al., loc. cit.; Sonnewald et al., loc. cit.; Bhattand Knowler, loc. cit.; Camirand et al., loc. cit.), sweet potato (Linet al., loc. cit., Lin et al., Plant Physiol. 95 (1991), 1250-1253) andrice (database accession number DDBJ No. D23280). Up to now, suchsequences are not known from maize.

Therefore, it is the object of the present invention to provide furthernucleic acid molecules encoding enzymes involved in starch biosynthesisand by means of which genetically modified plants may be produced thatshow an elevated or reduced activity of those enzymes, thereby promptinga modification in the chemical and/or physical properties of the starchsynthesized in these plants.

This object is achieved by the provision of the embodiments described inthe claims.

SUMMARY OF THE INVENTION

Therefore, the present invention relates to nucleic acid moleculesencoding proteins with the biological activity of a starch phosphorylasefrom maize, wherein such molecules preferably encode proteins whichcomprise the amino acid sequence depicted under Seq ID No. 2. Theinvention particularly relates to nucleic acid molecules which compriseall or part of the nucleotide sequence mentioned under Seq ID No. 1,preferably molecules, which comprise the coding region indicated in SeqID No. 1 or, as the case may be, corresponding ribonucleotide sequences.

The present invention further relates to nucleic acid molecules whichencode a starch phosphorylase from maize and one strand of whichhybridizes to one of the above-mentioned molecules. Nucleic acidmolecules that encode a starch phosphorylase from maize and the sequenceof which differs from the nucleotide sequences of the above-mentionedmolecules due to the degeneracy of the genetic code are also thesubject-matter of the invention.

The invention also relates to nucleic acid molecules showing a sequencewhich is complementary to the whole or to a part of one of theabove-mentioned sequences.

In this invention the term “hybridization” signifies hybridization underconventional hybridizing conditions, preferably under stringentconditions as described for example in Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). “Hybridization” preferablymeans that a hybridization takes place under the following conditions:

Hybridization buffer: 2×SSC; 10×Denhardt's solution (Fikoll 400+PEG+BSA;ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na₂HPO₄; 250 μg/ml herringsperm DNA; 50 μg/ml tRNA; or 0.25 M sodium phosphate buffer pH 7.2; 1 mMEDTA; 7% SDS

Hybridization temperature T=65 to 68° C.

Washing buffer: 0.2×SSC; 0.1% SDS

Washing temperature: T40 to 68° C.

Nucleic acid molecules hybridizing to the molecules of the invention mayprincipally encode starch phosphorylases from any desired maize plantexpressing such proteins.

Nucleic acid molecules hybridizing to the molecules according to theinvention may be isolated e.g. from genomic or from cDNA librariesproduced from maize plants or maize tissue. Alternatively, they may havebeen produced by means of recombinant DNA techniques or by means ofchemical synthesis. The identification and isolation of such nucleicacid molecules may take place by using the molecules according to theinvention or parts of these molecules or, as the case may be, thereverse complement strands of these molecules, e.g. by hybridizationaccording to standard methods (see e.g. Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

As a probe for hybridization e.g. nucleic acid molecules may be usedwhich exactly or basically contain the nucleotide sequences indicatedunder Seq ID No. 1 or parts thereof. The fragments used as hybridizationprobe may also be synthetic fragments which were produced by means ofthe conventional synthesizing methods and the sequence of which isbasically identical with that of a nucleic acid molecule according tothe invention.

The molecules hybridizing to the nucleic acid molecules of the inventionalso comprise fragments, derivatives and allelic variants of theabove-described nucleic acid molecules which encode a starchphosphorylase from maize as described in the invention. In this context,fragments are defined as parts of the nucleic acid molecules, which arelong enough in order to encode one of the described proteins. In thiscontext, the term derivatives means that the sequences of thesemolecules differ from the sequences of the above-mentioned nucleic acidmolecules at one or more positions and that they exhibit a high degreeof homology to these sequences. Homology means a sequence identity of atleast 40%, in particular an identity of at least 60%, preferably of morethan 80% and still more preferably a sequence identity of more than 90%.The deviations occurring when comparing with the above-described nucleicacid molecules might have been caused by deletion, substitution,insertion or recombination.

Moreover, homology means that functional and/or structural equivalenceexists between the respective nucleic acid molecules or the proteinsthey encode. The nucleic acid molecules, which are homologous to theabove-described molecules and represent derivatives of these molecules,are generally variations of these molecules, that constitutemodifications which exert the same biological function: These variationsmay be naturally occurring variations, for example sequences derivedfrom other maize varieties, or mutations, whereby these mutations mayhave occurred naturally or they may have been introduced by means of aspecific mutagenesis. Moreover the variations may be syntheticallyproduced sequences. The allelic variants may be naturally occurring aswell as synthetically produced variants or variants produced byrecombinant DNA techniques.

The proteins encoded by the various variants of the nucleic acidmolecules according to the invention exhibit certain commoncharacteristics. Enzyme activity, molecular weight, immunologicreactivity, conformation etc. may belong to these characteristics aswell as physical properties such as the mobility in gel electrophoresis,chromatographic characteristics, sedimentation coefficients, solubility,spectroscopic properties, stability, pH-optimum, temperature-optimumetc.

The enzymatic properties of starch phosphorylases were described above.The localization and the acitivity of the phosphorylase may be assessedas described, for example, in Steup and Latzko (Planta 145 (1979),69-75). The monomeric size may be determined by methods known to theskilled person.

The nucleic acid molecules of the invention may be DNA molecules,particularly cDNA or genomic molecules. The nucleic acid molecules ofthe invention may furthermore be RNA molecules. The nucleic acidmolecules of the invention may, e.g. be derived from natural sources orproduced by recombinant DNA techniques or synthetically.

Oligonucleotides hybridizing specifically to one of the nucleic acidmolecules of the invention are also subject-matter of the invention.Such oligonucleotides preferably have a length of at least 10,particularly of at least 15 and still more preferably have a length ofat least 50 nucleotides. They are characterized in that they hybridizespecifically to the nucleic acid molecules of the invention, i.e. theydo not or only to a small extent hybridize to nucleic acid sequencesencoding other proteins, particularly other starch phosphorylases. Theoligonucleotides of the invention may be used for example as primers fora PCR or as a hybridization probe for isolating related genes. They mayalso be components of antisense-constructs or DNA molecules encodingsuitable ribozymes.

Furthermore, the invention relates to vectors, especially plasmids,cosmids, viruses, bacteriophages and other vectors common in geneticengineering, which contain the above-mentioned nucleic acid molecules ofthe invention. Such vectors are preferably vectors which can be usedused for the transformation of plant cells. More preferably, they allowfor the integration of the nucleic acid molecules of the invention intothe genome of the plant cell, if necessary in combination with flankingregulatory regions. Examples are binary vectors which may be used in theAgrobacterium-mediated gene transfer.

In a preferred embodiment the nucleic acid molecules contained in thevectors are linked to regulatory elements that ensure the transcriptionand synthesis of a translatable RNA in procaryotic or eucaryotic cells.

The expression of the nucleic acid molecules of the invention inprocaryotic cells, e.g. in Escherichia coli, is interesting insofar asthis enables a more precise characterization of the enzymatic activitiesof the enzymes encoded by these molecules. In particular, it is possibleto characterize the product being synthesized by the respective enzymesin the absence of other enzymes which are involved in the starchsynthesis of the plant cell. This makes it possible to draw conclusionsabout the function, which the respective protein exerts during thestarch synthesis within the plant cell.

Moreover, it is possible to introduce various mutations into the nucleicacid molecules of the invention by means of conventionalmolecular-biological techniques (see e.g. Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which leads to thesynthesis of proteins with possibly modified biological properties. Bymeans of this it is on the one hand possible to produce deletionmutants, in which nucleic acid molecules are produced by continuingdeletions at the 5′- or the 3′-end of the encoding DNA-sequence. Thesenucleic acid molecules may lead to the synthesis of correspondinglyshortened proteins. Such deletions at the 5′-end of the nucleotidesequence make it possible, for example, to identify amino acid sequenceswhich are responsible for the translocation of the enzyme in theplastids (transit peptides). This allows for the specific production ofenzymes which due to the removal of the respective sequences are nolonger located in the plastids but within the cytosol, or which due tothe addition of other signal sequences are located in othercompartments.

On the other hand point mutations may also be introduced at positionswhere a modification of the amino acid sequence influences, for example,the enzyme activity or the regulation of the enzyme. In this way e.g.mutants with a modified K_(m)-value may be produced, or mutants whichare no longer subject to the regulation mechanisms by allostericregulation or covalent modification usually occurring in cells.

Furthermore, mutants may be produced exhibiting a modified substrate orproduct specificity. Moreover, mutants with a modifiedactivity-temperature-profile may be produced. For the geneticmanipulation in procaryotic cells the nucleic acid molecules of theinvention or parts of these molecules may be integrated into plasmidswhich allow for a mutagenesis or a sequence modification byrecombination of DNA sequences. By means of standard methods (cf.Sambrook et al., 1989, Molecular Cloning: A laboratory manual, 2ndedition, Cold Spring Harbor Laboratory Press, NY, USA) base exchangesmay be carried out or natural or synthetic sequences may be added. Inorder to connect the DNA fragments, adapters or linkers may be attachedto the fragments. Moreover, use can be made of manipulations which offersuitable restriction sites or which remove superfluous DNA orrestriction sites. Wherever use is made of insertions, deletions orsubstitutions, in vitro mutagenesis, “primer repair”, restriction orligation may be used. For analyzing use is usually made of a sequenceanalysis, a restriction analysis or furtherbiochemico-molecularbiological methods.

In a further embodiment the invention relates to host cells, inparticular procaryotic or eucaryotic cells, which have been transformedby an above-mentioned nucleic acid molecule of the invention or by avector of the invention, as well as cells derived from cells transformedin such a way and containing a nucleic acid molecule of the invention ora vector of the invention. This is preferably a bacterial cell or aplant cell.

Furthermore, the proteins encoded by the nucleic acid molecules of theinvention are the subject-matter of the invention as well as methods fortheir production in which a host cell of the invention is cultivatedunder conditions that allow for the synthesis of the protein and inwhich the protein is subsequently isolated from the cultivated cellsand/or the culture medium.

By making available the nucleic acid molecules of the invention it isnow possible—by means of recombinant DNA techniques—to interfere withthe starch metabolism of plants in a way so far impossible. Thereby, thestarch metabolism may be modified in such a way that a modified starchis synthesized which e.g. is modified, compared to the starchsynthesized in wildtype plants, with respect to its physico-chemicalproperties, especially the amylose/amylopectin ratio, the degree ofbranching, the average chain length, the phosphate content, thepastification behavior, the size and/or the shape of the starch granule,the viscuous properties and/or the side chain distribution. There is thepossibility of increasing the yield of genetically modified plants byincreasing the activity of the proteins of the invention, e.g. byoverexpressing the respective nucleic acid molecules or by makingmutants available which are no longer subject to cell-specificregulation schemes and/or different temperature-dependencies withrespect to their activity. The economic significance of the chance tointerfere with the starch synthesis of maize alone is obvious: maize isthe world's most important plant with regard to the production ofstarch. 80% of the starch globally produced each year is derived frommaize.

Therefore it is possible to express the nucleic acid molecules of theinvention in plant cells in order to increase the activity of therespective starch phosphorylases. Furthermore, the nucleic acidmolecules of the invention may be modified by means of methods known tothe skilled person, in order to produce starch phosphorylases accordingto the invention which are no longer subject to the cell-specificregulation mechanisms or show modified temperature-dependencies orsubstrate or product specificities.

In expressing the nucleic acid molecules of the invention in plants thesynthesized proteins may in principle be located in any desiredcompartment within the plant cell. In order to locate it within aspecific compartment, the sequence ensuring the localization in theplastids must be deleted and the remaining coding region optionally hasto be linked to DNA sequences which ensure localization in therespective compartment. Such sequences are known (see e.g. Braun et al.,EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA85 (1988), 846-850; Sonnewald et al., Plant J. 1 (1991), 95-106).

Thus, the present invention also relates to transgenic plant cellstransformed with a nucleic acid molecule or a vector of the invention,as well as it relates to transgenic plant cells which are derived fromcells transformed in such a way. Such cells contain a nucleic acidmolecule of the invention which is preferably linked to regulatory DNAelements ensuring the transcription in plant cells, especially with apromoter. Such cells differ from naturally occurring plant cells, e.g.in that they contain a nucleic acid molecule of the invention which doesnot naturally occur in such cells or in that such a molecule isintegrated at some position in the genome of the cell at which it doesnot naturally occur, i.e. in a different genomic environment. Moreover,such transgenic plant cells of the invention differ from naturallyoccurring plants among other things in that at least one copy of thenucleic acid molecule of the invention is stably integrated in theirgenome, possibly in addition to the naturally occurring copies. If thenucleic acid molecule(s) integrated into the cell(s) is/are (an)additional copy (copies) of molecules already occurring naturally in thecells, the plant cells of the invention differ from the naturallyoccurring plant cells particularly in that this/these additionalcopy/copies is/are integrated at a location in the genome at which theydo not occur naturally. This may be proved, for example, by means of aSouthern Blot analysis.

Furthermore, the plant cells of the invention differ from naturallyoccurring plant cells preferably in at least one of the followingfeatures: if the introduced nucleic acid molecule of the invention isheterologous with regard to the plant cell, the transgenic plant cellscomprise transcripts of the introduced nucleic acid molecules of theinvention. This may be determined, for example, by means of a NorthernBlot analysis. The plant cells of the invention preferably contain aprotein encoded by an introduced nucleic acid molecule of the invention.This may be determined, for example, by means of immunological methods,in particular by means of a Western Blot analysis.

If the introduced nucleic acid molecule of the invention is homologouswith regard to the plant cell, the cells of the invention may bedistinguished from naturally occurring cells, for example, by theadditional expression of nucleic acid molecules of the invention.

The transgenic plant cells of the invention preferably contain moretranscripts of the nucleic acid molecules of the invention. This may beshown, for example, by Northern Blot analysis. Thereby, “more”preferably means at least 10% more, more preferably at least 20% moreand particularly preferred at least 50% more transcripts than thecorresponding non-transformed cells. Furthermore, the cells preferablycomprise a corresponding increase in the amount of the protein of theinvention (at least 10%, 20% or, as the case may be, 50%). Thetransgenic plant cells may be regenerated to whole plants according tomethods known to the skilled person.

The plants obtained by regenerating the transgenic plant cells of theinvention are also the subject-matter of the present invention. Afurther subject-matter of the invention are plants which contain theabove-described transgenic plant cells. The transgenic plants may inprinciple be plants of any desired species, i.e. they may bemonocotyledonous as well as dicotyledonous plants. These are preferablyuseful plants, i.e. plants cultivated by man as foodstuffs or fortechnical, in particular for industrial purposes. They are in particularstarch-synthesizing or starch-storing plants such as cereals (rye,barley, oats, wheat, millet, sago etc.), amaranth (Amaranthus), rice,lentil, peas, chick-pea, mung bean, broad bean, scarlet runner bean,cassava, potato, sweet potato, tomato, rape seed, soy bean, hemp, flax,sunflower, cow pea or arrowroot. Maize is particularly preferred.

The invention also relates to propagation material of the plants of theinvention, e.g. fruits, seeds, tubers, root-stocks, seedlings, cuttings,calli, protoplasts, cell cultures etc.

The present invention further relates to a method for producing amodified starch comprising the step of extracting the starch from anabove-described plant of the invention and/or from starch-storing partsof such a plant. Preferably, such a method also comprises the step ofharvesting the cultivated plants and/or starch-storing parts of suchplants before extracting the starch. Most preferably, it furthercomprises the step of cultivating the plants of the invention beforeharvesting. Methods for the extraction of starch from plants or fromstarch-storing parts of plants are known to the skilled person. Methodsfor the extraction of starch from maize seeds have been described e.g.in Eckhoff et al. (Cereal Chem. 73 (1996) 54-57). The extraction ofmaize starch on an industrial level is usually achieved by the so-calledwet-milling technique. Furthermore, methods for the extraction of starchfrom various other starch-storing plants have been described, e.g. in“Starch: Chemistry and Technology (Editor: Whistler, BeMiller andPaschall (1994), 2^(nd) edition, Academic Press Inc. London Ltd; ISBN0-12-746270-8; see e.g. chapter XII, page 412-468: maize and sorghumstarches: production; by Watson; chapter XIII, page 469-479: tapioca,arrowroot and sago starches: production; by Corbishley and Miller;chapter XIV, page 479-490: potato starch: production and use; by Mitch;chapter XV, page 491 to 506: wheat starch: production, modification anduse; by Knight and Oson; and chapter XVI, page 507 to 528: rice starch:production and use; by Rohmer and Klem). Appliances generally used forextracting starch from plant material are separators, decanters,hydrocyclones, spray dryers and cyclon driers.

Due to the expression or, as the case may be, additional expression of anucleic acid molecule of the invention, the transgenic plant cells andplants described in the invention synthesize a starch which compared tostarch synthesized in wildtype plants is modified for example in itsphysico-chemical properties, in particular in the amylose/amylopectinratio, the degree of branching, the average chain-length, thephosphate-content, the pastification behavior, the size and/or the shapeof the starch granule. Compared with wildtype-starch, such starch may bemodified in particular with respect to its viscosity and/or the gelformation properties of the glues of this starch.

Thus, also the starch obtainable from transgenic plant cells, plants aswell as from the propagation material according to the invention is thesubject-matter of the present invention.

By means of the nucleic acid molecules of the invention it isfurthermore possible to produce maize plant cells and maize plants inwhich the activity of a protein of the invention is reduced. This alsoleads to the synthesis of a starch with modified chemical and/orphysical properties when compared to the starch from wildtype plantcells.

Thus, transgenic maize plant cells, in which the activity of a proteinaccording to the invention is reduced when compared to non-transformedcells, are a further subject-matter of the invention.

The production of maize plant cells with a reduced activity of a proteinof the invention may for example be achieved by the expression of acorresponding antisense-RNA, of a sense-RNA for achieving a cosupressioneffect or the expression of a correspondingly constructed ribozyme,which specifically cleaves transcripts encoding one of the proteins ofthe invention, using the nucleic acid molecules of the invention. Inorder to reduce the activity of a protein of the invention preferablyantisense-RNA is expressed in plant cells.

In order to express an antisense-RNA, on the one hand DNA molecules canbe used which comprise the complete sequence encoding a protein of theinvention, including possibly existing flanking sequences as well as DNAmolecules, which only comprise parts of the coding sequence wherebythese parts have to be long enough in order to prompt anantisense-effect within the cells. Basically, sequences with a minimumlength of 15 bp, preferably with a length of 100-500 bp, and for anefficient antisense-inhibition, in particular sequences with a length ofmore than 500 bp may be used. Generally DNA-molecules are used which areshorter than 5000 bp, preferably sequences with a length of less than2500 bp.

Use may also be made of DNA sequences which are highly homologous, butnot completely identical to the sequences of the DNA molecules of theinvention. The minimal homology should be more than about 65%.Preferably, use should be made of sequences with homologies between 95and 100%.

Alternatively, the reduction of the enzyme activity of the starchphosphorylase in plant cells may also be achieved by means of acosuppression effect, as indicated above. The method is known to theskilled person and has been described, for example, in Jorgensen (TrendsBiotechnol. 8 (1990), 340-344), Niebel et al. (Curr. Top. Microbiol.Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top. Microbiol.Immunol. 197 (1995), 43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29(1995), 149-159), Vaucheret et al. (Mol. Gen. Genet. 248 (1995),311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621) andother sources.

Thus, a subject matter of the present invention are in particulartransgenic maize plant cells

(a) comprising a DNA molecule which may lead to the synthesis of anantisense RNA which leads to the reduction of the expression of nucleicacid molecules of the invention; and/or

(b) comprising a DNA molecule which may lead to the synthesis of acosupression RNA which leads to the reduction of the expression ofnucleic acid molecules of the invention; and/or

(c) comprising a DNA molecule which may lead to the synthesis of aribozyme which specifically cleaves transcripts of nucleic acidmolecules of the invention.

The cells of the invention preferably show a reduction in the amount oftranscripts encoding a protein of the invention when compared tocorresponding non-transformed cells, whereby the reduction is preferablyat least 30%, more preferably at least 50%, even more preferably atleast 70% and most preferably at least 90%. The amount of transcripts inthe cells may, for example, be determined by means of a Northern Blotanalysis. The cells preferably show a corresponding, i.e. at least 30%,50%, 70% or 90% reduction in the amount of the protein of the inventionwhen compared to non-transformed cells. The amount of proteins may bedetermined, for example, by means of immunological methods, such asWestern Blot analysis.

Maize plants containing the transgenic maize plant cells of theinvention are also the subject matter of the invention. The inventionalso relates to the propagation material of the plants of the invention,in particular to seeds, calli, protoplasts, cell cultures etc.

The present invention further relates to a method for producing amodified starch comprising the step of extracting the starch from anabove-described plant of the invention and/or from starch-storing partsof such a plant. Preferably, such a method also comprises the step ofharvesting the cultivated plants and/or starch-storing parts of suchplants before extracting the starch. Most preferably, it furthercomprises the step of cultivating the plants of the invention beforeharvesting.

Starch obtainable from the aforementioned transgenic maize plant cells,maize plants as well as propagation material is a further subject matterof the invention as well as starch obtainable from the above-describedmethod of the invention. Due to the reduction of the activity of aprotein of the invention, the transgenic maize plant cells and maizeplants synthesize a starch which compared to starch synthesized inwildtype plants is modified, for example, in its physico-chemicalproperties, in particular in the amylose/amylopectin ratio, the degreeof branching, the average chain-length, the phosphate-content, thepastification behavior, the side-chain distribution, the size and/or theshape of the starch granule. Compared with wildtype-starch, such starchmay be modified in particular with respect to its viscosity and/or thegel formation properties of the glues of this starch.

The starches of the invention may be modified according to techniquesknown to the skilled person; in unmodified as well as in modified formthey are suitable for the use in foodstuffs and for the use innon-foodstuffs.

Basically, the possibilities of uses of the starch can be subdividedinto two major fields. One field comprises the hydrolysis products ofstarch, essentially glucose and glucans components obtained by enzymaticor chemical processes. They can be used as starting material for furtherchemical modifications and processes, such as fermentation. In thiscontext, it might be of importance that the hydrolysis process can becarried out simply and inexpensively. Currently, it is carried outsubstantially enzymatically using amyloglucosidase. It is thinkable thatcosts might be reduced by using lower amounts of enzymes for hydrolysisdue to changes in the starch structure, e.g. increasing the surface ofthe grain, improved digestibility due to less branching or a stericstructure, which limits the accessibility for the used enzymes. Theother field in which the starch is used because of its polymer structureas so-called native starch, can be subdivided into two further areas:

1. Use in foodstuffs

Starch is a classic additive for various foodstuffs, in which itessentially serves the purpose of binding aqueous additives and/orcauses an increased viscosity or an increased gel formation. Importantcharacteristic properties are flowing and sorption behavior, swellingand pastification temperature, viscosity and thickening performance,solubility of the starch, transparency and paste structure, heat, shearand acid resistance, tendency to retrogradation, capability of filmformation, resistance to freezing/thawing, digestibility as well as thecapability of complex formation with e.g. inorganic or organic ions.

A preferred area of application of native starch is the field ofbakery-goods and pasta.

2. Use in non-foodstuffs

The other major field of application is the use of starch as an adjuvantin various production processes or as an additive in technical products.The major fields of application for the use of starch as an adjuvantare, first of all, the paper and cardboard industry. In this field, thestarch is mainly used for retention (holding back solids), for sizingfiller and fine particles, as solidifying substance and for dehydration.In addition, the advantageous properties of starch with regard tostiffness, hardness, sound, grip, gloss, smoothness, tear strength aswell as the surfaces are utilized.

2.1 Paper and cardboard industry

Within the paper production process, a differentiation can be madebetween four fields of application, namely surface, coating, mass andspraying.

The requirements on starch with regard to surface treatment areessentially a high degree of brightness, corresponding viscosity, highviscosity stability, good film formation as well as low formation ofdust. When used in coating the solid content, a corresponding viscosity,a high capability to bind as well as a high pigment affinity play animportant role. As an additive to the mass rapid, uniform, loss-freedispersion, high mechanical stability and complete retention in thepaper pulp are of importance. When using the starch in spraying,corresponding content of solids, high viscosity as well as highcapability to bind are also significant.

2.2 Adhesive industry

A major field of application is, for instance, in the adhesive industry,where the fields of application are subdivided into four areas: the useas pure starch glue, the use in starch glues prepared with specialchemicals, the use of starch as an additive to synthetic resins andpolymer dispersions as well as the use of starches as extenders forsynthetic adhesives. 90% of all starch-based adhesives are used in theproduction of corrugated board, paper sacks and bags, compositematerials for paper and aluminum, boxes and wetting glue for envelopes,stamps, etc.

2.3 Textile and textile care industry

Another possible use as adjuvant and additive is in the production oftextiles and textile care products. Within the textile industry, adifferentiation can be made between the following four fields ofapplication: the use of starch as a sizing agent, i.e. as an adjuvantfor smoothing and strengthening the burring behavior for the protectionagainst tensile forces active in weaving as well as for the increase ofwear resistance during weaving, as an agent for textile improvementmainly after quality-deteriorating pretreatments, such as bleaching,dying, etc., as thickener in the production of dye pastes for theprevention of dye diffusion and as an additive for warping agents forsewing yarns.

2.4 Building industry

The fourth area of application of starch is its use as an additive inbuilding materials. One example is the production of gypsum plasterboards, in which the starch mixed in the thin plaster pastifies with thewater, diffuses at the surface of the gypsum board and thus binds thecardboard to the board. Other fields of application are admixing it toplaster and mineral fibers. In ready-mixed concrete, starch may be usedfor the deceleration of the sizing process.

2.5 Ground stabilization

Furthermore, the starch is advantageous for the production of means forground stabilization used for the temporary protection of groundparticles against water in artificial earth shifting. According tostate-of-the-art knowledge, combination products consisting of starchand polymer emulsions can be considered to have the same erosion- andincrustation-reducing effect as the products used so far; however, theyare considerably less expensive.

2.6 Use of starch in plant protectives and fertilizers

Another field of application is the use of starch in plant protectivesfor the modification of the specific properties of these preparations.For instance, starches are used for improving the wetting of plantprotectives and fertilizers, for the dosed release of the activeingredients, for the conversion of liquid, volatile and/or odorousactive ingredients into microcristalline, stable, deformable substances,for mixing incompatible compositions and for the prolongation of theduration of the effect due to a reduced disintegration.

2.7 Drugs, medicine and cosmetics industry

Starch may also be used in the fields of drugs, medicine and in thecosmetics industry. In the pharmaceutical industry, the starch may beused as a binder for tablets or for the dilution of the binder incapsules. Furthermore, starch is suitable as disintegrant for tabletssince, upon swallowing, it absorbs fluid and after a short time itswells so much that the active ingredient is released. For qualitativereasons, medicinal flowance and dusting powders are further fields ofapplication. In the field of cosmetics, the starch may for example beused as a carrier of powder additives, such as scents and salicylicacid. A relatively extensive field of application for the starch istoothpaste.

2.8 Starch as an additive in coal and briquettes

The use of starch as an additive in coal and briquettes is alsothinkable. By adding starch, coal can be quantitatively agglomeratedand/or briquetted in high quality, thus preventing prematuredisintegration of the briquettes. Barbecue coal contains between 4 and6% added starch, calorated coal between 0.1 and 0.5%. Furthermore, thestarch is suitable as a binding agent since adding it to coal andbriquette can considerably reduce the emission of toxic substances.

2.9 Processing of ore and coal slurry

Furthermore, the starch may be used as a flocculant in the processing ofore and coal slurry.

2.10 Starch as an additive in casting

Another field of application is the use as an additive to processmaterials in casting. For various casting processes cores produced fromsands mixed with binding agents are needed. Nowadays, the most commonlyused binding agent is bentonite mixed with modified starches, mostlyswelling starches.

The purpose of adding starch is increased flow resistance as well asimproved binding strength. Moreover, swelling starches may fulfill moreprerequisites for the production process, such as dispersability in coldwater, rehydratisability, good mixability in sand and high capability ofbinding water.

2.11 Use of starch in rubber industry

In the rubber industry starch may be used for improving the technicaland optical quality. Reasons for this are improved surface gloss, gripand appearance. For this purpose, the starch is dispersed on the stickyrubberized surfaces of rubber substances before the cold vulcanization.It may also be used for improving the printability of rubber.

2.12 Production of leather substitutes

Another field of application for the modified starch is the productionof leather substitutes.

2.13 Starch in synthetic polymers

In the plastics market the following fields of application are emerging:the integration of products derived from starch into the processingprocess (starch is only a filler, there is no direct bond betweensynthetic polymer and starch) or, alternatively, the integration ofproducts derived from starch into the production of polymers (starch andpolymer form a stable bond).

The use of the starch as a pure filler cannot compete with othersubstances such as talcum. This situation is different when the specificstarch properties become effective and the property profile of the endproducts is thus clearly changed. One example is the use of starchproducts in the processing of thermoplastic materials, such aspolyethylene. Thereby, starch and the synthetic polymer are combined ina ratio of 1:1 by means of coexpression to form a ‘master batch’, fromwhich various products are produced by means of common techniques usinggranulated polyethylene. The integration of starch in polyethylene filmsmay cause an increased substance permeability in hollow bodies, improvedwater vapor permeability, improved antistatic behavior, improvedanti-block behavior as well as improved printability with aqueous dyes.Another possibility is the use of the starch in polyurethane foams. Dueto the adaptation of starch derivatives as well as due to theoptimization of processing techniques, it is possible to specificallycontrol the reaction between synthetic polymers and the starch's hydroxygroups. The results are polyurethane films having the following propertyprofiles due to the use of starch: a reduced coefficient of thermalexpansion, decreased shrinking behavior, improved pressure/tensionbehavior, increased water vapor permeability without a change in wateracceptance, reduced flammability and cracking density, no drop off ofcombustible parts, no halides and reduced aging. Disadvantages thatpresently still exist are reduced pressure and impact strength.

Product development of film is not the only option. Also solid plasticsproducts, such as pots, plates and bowls can be produced by means of astarch content of more than 50%. Furthermore, the starch/polymermixtures offer the advantage that they are much easier biodegradable.

Furthermore, due to their extreme capability to bind water, starch graftpolymers have gained utmost importance. These are products having abackbone of starch and a side lattice of a synthetic monomer grafted onaccording to the principle of radical chain mechanism. The starch graftpolymers available nowadays are characterized by an improved binding andretaining capability of up to 1000 g water per g starch at a highviscosity. These super absorbers are used mainly in the hygiene field,e.g. in products such as diapers and sheets, as well as in theagricultural sector, e.g. in seed pellets.

What is decisive for the use of the new starch modified by recombinantDNA techniques are, on the one hand, structure, water content, proteincontent, lipid content, fiber content, ashes/phosphate content,amylose/amylopectin ratio, distribution of the relative molar mass,degree of branching, granule size and shape as well as crystallization,and on the other hand, the properties resulting in the followingfeatures: flow and sorption behavior, pastification temperature,viscosity, thickening performance, solubility, paste structure,transparency, heat, shear and acid resistance, tendency toretrogradation, capability of gel formation, resistance tofreezing/thawing, capability of complex formation, iodine binding, filmformation, adhesive strength, enzyme stability, digestibility andreactivity.

The production of modified starch by genetically operating with atransgenic plant may modify the properties of the starch obtained fromthe plant in such a way as to render further modifications by means ofchemical or physical methods superfluous. On the other hand, thestarches modified by means of recombinant DNA techniques might besubjected to further chemical modification, which will result in furtherimprovement of the quality for certain of the above-described fields ofapplication. These chemical modifications are principally known to theperson skilled in the art. These are particularly modifications by meansof

heat treatment

acid treatment

oxidation and

esterification leading to the formation of phosphate, nitrate, sulfate,xanthate, acetate and citrate starches. Other organic acids may also beused for the esterification:

formation of starch ethers starch alkyl ether, O-allyl ether,hydroxylalkyl ether, O-carboxylmethyl ether, N-containing starch ethers,P-containing starch ethers and S-containing starch ethers.

formation of branched starches

formation of starch graft polymers.

In order to express the nucleic acid molecules of the invention insense- or antisense-orientation in plant cells, these are normallylinked to regulatory DNA elements which ensure the transcription inplant cells. Such regulatory DNA elements are particularly promoters.Basically any promoter which is active in plant cells may be used forthe expression.

The promoter may be selected in such a way that the expression takesplace constitutively or in a certain tissue, at a certain point of timeof the plant development or at a point of time determined by externalcircumstances. With respect to the plant the promoter may be homologousor heterologous. Suitable promoters for a constitutive expression are,e.g. the 35S RNA promoter of the Cauliflower Mosaic Virus and theubiquitin promoter from maize. For a tuber-specific expression inpotatoes the patatin gene promoter B33 (Rochα-Sosa et al., EMBO J. 8(1989), 23-29) can be used. A promoter which ensures expression only inphotosynthetically active tissues is, e.g. the ST-LS1 promoter(Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947;Stockhaus et al., EMBO J. 8 (1989), 2445-2451). For anendosperm-specific expression the HMG promoter from wheat, the USPpromoter, the phaseolin promoter or promoters from zein genes from maizeare suitable. Furthermore, a termination sequence may exist which servesto correctly end the transcription and to add a poly-A-tail to thetranscript which is believed to stabilize the transcripts. Such elementsare described in the literature (cf. Gielen et al., EMBO J. 8 (1989),23-29) and can be exchanged as desired.

The present invention provides nucleic acid molecules encoding a newtype of starch phosphorylase identified in maize. This allows for theidentification of the function of this starch phosphorylase in thestarch biosynthesis as well as for the production of geneticallymodified plants in which the activity of this enzyme is modified. Thisenables the synthesis of starch with a modified structure and thereforewith modified physico-chemical properties in the plants manipulated insuch a way.

Principally, the nucleic acid molecules of the invention may also beused in order to produce plants in which the activity of the starchphosphorylase of the invention is elevated or reduced and in which atthe same time the activities of other enzymes involved in the starchbiosynthesis are modified. Thereby, all kinds of combinations andpermutations are thinkable. By modifying the activities of a starchphosphorylase in plants, a synthesis of a starch modified in itsstructure is brought about. Moreover, nucleic acid molecules encoding aprotein of the invention, or corresponding antisense-constructs may beintroduced into the plant cells, in which the synthesis of endogenousGBSS I-, SSS- or GBSS II-proteins is already inhibited due to anantisense-effect or a mutation, or in which the synthesis of thebranching enzyme is inhibited (as described e.g. in WO92/14827 or in theae-mutant (Shannon and Garwood, 1984, in Whistler, BeMiller andPaschall, Starch: Chemistry and Technology, Academic Press, London,2^(nd) Edition: 25-86)).

If the inhibition of the synthesis of several enzymes involved in thestarch biosynthesis in transformed plants is to be achieved, DNAmolecules can be used for transformation, which at the same time containseveral regions in antisense-orientation controlled by a suitablepromoter and encoding the corresponding enzymes. Hereby, each sequencemay be controlled by its own promoter or else the sequences may betranscribed as a fusion from a common promoter. The last alternativewill generally be preferred as in this case the synthesis of therespective proteins should be inhibited to approximately the sameextent. For the length of the single coding regions used in such aconstruct the same applies which has already been said above inconnection with the production of antisense-constructs. There is noupper limit for the amount of the antisense fragments transcribed by apromoter in such a DNA molecule. The produced transcript, however,should usually not be longer than 10 kb or, preferably, 5 kb.

Coding regions which are localized in such DNA molecules in combinationwith other coding regions in antisense orientation behind a suitablepromoter may be derived from DNA sequences coding for the followingproteins: starch granule-bound (GBSS I and II) and soluble starchsynthases (SSS I and II), branching enzymes, debranching enzymes anddisproportioning enzymes. This enumeration only serves as an example.The use of other DNA sequences is also thinkable within the framework ofsuch a combination.

By means of such constructs it is possible to simultaneously inhibit thesynthesis of a number of enzymes in plant cells transformed therewith.

Furthermore, the constructs may be inserted into classical mutants whichare deficient for at least one gene of the starch biosynthesis (Shannonand Garwood, 1984, in Whistler, BeMiller and Paschall, Starch: Chemistryand Technology, Academic Press, London, 2nd edition: 25-86). Thesedeficiencies may relate to the following proteins: starch granule-bound(GBSS I and II) and soluble starch synthases (SSS I and II), branchingenzymes (BE I and II), debranching enzymes (R enzymes), disproportioningenzymes and starch phosphorylases. This enumeration only serves as anexample.

By proceeding in such a way it is furthermore possible to simultaneouslyinhibit the synthesis of a number of enzymes in plant cells transformedtherewith.

In order to prepare the introduction of foreign genes into higher plantsa multitude of cloning vectors is available comprising a replicationsignal for E.coli and a marker gene for the selection of transformedbacterial cells. Examples for such vectors are pBR322, pUC series, M13mpseries, pACYC184 etc. The desired sequence may be integrated into thevector at a suitable restriction site. The obtained plasmid ispreferably used for the transformation of E.coli cells. TransformedE.coli cells are cultivated in a suitable medium and subsequentlyharvested and lysed. The plasmid is recovered. As an analyzing methodfor the characterization of the obtained plasmid DNA use is generallymade of restriction analyses, gel electrophoreses and otherbiochemico-molecularbiological methods. After each manipulation theplasmid DNA may be cleaved and the obtained DNA fragments may be linkedto other DNA sequences. Each plasmid DNA sequence may be cloned into thesame or in other plasmids.

In order to introduce DNA into plant host cells a wide range oftechniques are at disposal. These techniques comprise the transformationof plant cells with T-DNA by using Agrobacterium tumefaciens orAgrobacterium rhizogenes as transformation medium, the fusion ofprotoplasts, the injection and the electroporation of DNA, theintegration of DNA by means of the biolistic method as well as furtherpossibilities. In the case of injection and electroporation of DNA intoplant cells, there are no special demands made to the plasmids used.Simple plasmids such as pUC derivatives may be used. However, in casethat whole plants are to be regenerated from cells transformed in such away, a selectable marker gene should be present.

Depending on the method of introducing desired genes into the plantcell, further DNA sequences may be necessary. If the Ti- or Ri-plasmidis used e.g. for the transformation of the plant cell, in general atleast the right border, more frequently, however, the right and leftborder of the Ti- and Ri-plasmid T-DNA should be connected to theforeign gene to be introduced as a flanking region.

If Agrobacteria are used for the transformation, the DNA which is to beintroduced should advantageously be cloned into special plasmids, namelyeither into an intermediate vector or into a binary vector. Due tosequences homologous to the sequences within the T-DNA, the intermediatevectors may be integrated into the Ti- or Ri-plasmid of theAgrobacterium due to homologous recombination. This also contains thevir-region necessary for the transfer of the T-DNA. Intermediate vectorscannot replicate in Agrobacteria. By means of a helper plasmid theintermediate vector may be transferred to Agrobacterium tumefaciens(conjugation). Binary vectors may replicate in E.coli as well as inAgrobacteria. They contain a selectable marker gene as well as a linkeror polylinker which is framed by the right and the left T-DNA borderregion. They may be transformed directly into the Agrobacteria (Holsterset al. Mol. Gen. Genet. 163 (1978), 181-187). The Agrobacterium actingas host cell should contain a plasmid carrying a vir-region. Thevir-region is necessary for the transfer of the T-DNA into the plantcell. Additional T-DNA may be present. The Agrobacterium transformed insuch a way is used for the transformation of plant cells.

The use of T-DNA for the transformation of plant cells was investigatedintensely and described sufficiently in EP 120 516; Hoekema, In: TheBinary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam(1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and Anet al. EMBO J. 4 (1985), 277-287.

For transferring the DNA into the plant cells, plant explants maysuitably be co-cultivated with Agrobacterium tumefaciens orAgrobacterium rhizogenes. From the infected plant material (e.g. piecesof leaves, stem segments, roots, but also protoplasts orsuspension-cultivated plant cells) whole plants may then be regeneratedin a suitable medium which may contain antibiotics or biozides for theselection of transformed cells. The plants obtained in such a way maythen be examined as to whether the integrated DNA is present or not.Other possibilities in order to integrate foreign DNA by using thebiolistic method or by transforming protoplasts are known to the skilledperson (cf. e.g. Willmitzer, L., 1993 Transgenic plants. In:Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G.Reed, A. Pühler, P. Stadler, editors), Vol. 2, 627-659, VCH Weinheim-NewYork-Basel-Cambridge). Whereas the transformation of dicotyledonousplants by Ti-plasmid-vector systems by means of Agrobacteriumtumefaciens is a well-established method, more recent studies indicatethat the transformation with vectors based on Agrobacterium can also beused in the case of monocotyledonous plants (Chan et al., Plant Mol.Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282).

Alternative systems for the transformation of monocotyledonous plantsare the transformation by means of the biolistic approach, protoplasttransformation, electroporation of partially permeablized cells, theintroduction of DNA by means of glass fibers.

There are various references in the relevant literature dealingspecifically with the transformation of maize (cf. e.g. WO95/06128, EP 0513 849; EP 0 465 875). In EP 292 435 a method is described by means ofwhich fertile plants may be obtained starting from mucousless, friablegranulous maize callus. In this context it was furthermore observed byShillito et al. (Bio/Technology 7 (1989), 581) that for regeneratingfertile plants it is necessary to start from callus-suspension culturesfrom which a culture of dividing protoplasts can be produced which iscapable to regenerate to plants. After an in vitro cultivation period of7 to 8 months Shillito et al. obtain plants with viable descendantswhich, however, exhibited abnormalities in morphology andreproductivity.

Prioli and Söndahl (Bio/Technology 7 (1989), 589) have described how toregenerate and to obtain fertile plants from maize protoplasts of theCateto maize inbreed Cat 100-1. The authors assume that the regenerationof protoplast to fertile plants depends on a number of various factorssuch as the genotype, the physiological state of the donor-cell and thecultivation conditions. Once the introduced DNA has been integrated inthe genome of the plant cell, it usually continues to be stable thereand also remains within the descendants of the originally transformedcell. It usually contains a selectable marker which confers resistanceagainst biozides or against an antibiotic such as kanamycin, G 418,bleomycin, hygromycin or phosphinotricine etc. to the transformed plantcells. The individually selected marker should therefore allow for aselection of transformed cells against cells lacking the introduced DNA.

The transformed cells grow in the usual way within the plant (see alsoMcCormick et al., Plant Cell Reports 5 (1986), 81-84). The resultingplants can be cultivated in the usual way and cross-bred with plantshaving the same transformed genetic heritage or another geneticheritage. The resulting hybrid individuals have the correspondingphenotypic properties.

Two or more generations should be grown in order to ensure whether thephenotypic feature is kept stably and whether it is transferred.Furthermore, seeds should be harvested in order to ensure that thecorresponding phenotype or other properties will remain.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a construct for antisense inhibition of a plastidic isoformof starch phosphorylase in maize.

DETAILED DESCRIPTION OF THE INVENTION

The examples illustrate the invention.

Media and solutions used in the examples:

20 × SSC: 175.3 g NaCl 88.2 g sodium citrate ad 1000 ml with ddH₂O pH7.0 with 10 N NaOH YT 8 g Bacto-Yeast extract 5 g Bacto-Tryptone 5 gNaCl ad 1000 ml with ddH₂O Protoplast isolation medium (100 ml)Cellulase Onozuka R S 800 mg (Meiji Seika, Japan) Pectolyase Y 23 40 mgKNO₃ 200 mg KH₂PO₄ 136 mg K₂HPO₄ 47 mg CaCl₂ 2H₂O 147 mg MgSO₄ 7H₂O 250mg Bovine serum albumine (BSA) 20 mg Glucose 4000 mg Fructose 4000 mgSucrose 1000 mg pH 5.8 Osmolarity 660 mosm.

Protoplast washing solution 1: like protoplast isolating solution, butwithout cellulase, pectolyase and BSA

Transfomation buffers: a) Glucose 0.5 M MES 0.1 % MgCl₂ 6H₂O 25 mM pH5.8 adjust to 600 mosm. b) PEG 6000-solution Glucose 0.5 M MgCl₂ 6H₂O100 mM Hepes 20 mM pH 6.5

PEG 6000 is added to the buffer described in b) immediately prior to theuse of the solution (40% w/v PEG). The solution is filtered with a 0.45μm sterile filter.

W5 solution CaCl₂ 125 mM NaCl 150 mM KCl 5 mM Glucose 50 mM Protoplastculture medium (indicated in mg/l) KNO₃ 3000 (NH₄)₂SO₄ 500 MgSO₄ 7H₂O350 KH₂PO₄ 400 CaCl₂ 2H₂O 300

Fe-EDTA and trace elements as in the Murashige-Skoog medium (Physiol.Plant, 15 (1962), 473).

m-inosite 100 Thiamine HCl 1.0 Nicotine acid amide 0.5 Pyridoxine HCl0.5 Glycine 2.0 Glucuronic acid 750 Galacturonic acid 750 Galactose 500Maltose 500 Glucose 36,000 Fructose 36,000 Sucrose 30,000 Asparagine 500Glutamine 100 Proline 300 Caseinhydrolysate 500 2,4 dichlorophenoxyacetic acid (2,4-D) 0.5 pH 5.8 Osmolarity 600 mosm

In the examples the following methods were used:

1. Cloning methods

For cloning in E.coli the vector pBluescript II SK (Stratagene) wasused.

2. Bacterial strains

For the Bluescript vector and for the pUSP constructs use was made ofthe E.coli strain DH5α (Bethesda Research Laboratories, Gaithersburgh,USA). The E.coli strain XL1-Blue was used for in vivo excision.

3. Transformation of maize

(a) Production of protoplasts of the cell line DSM 6009

Protoplast isolation

2-4 days, preferably 3 days after the last change of medium in aprotoplast suspension culture the liquid medium is pumped off and theremaining cells are washed in 50 ml protoplast washing solution 1 andsucked dry once more. 10 ml protoplast isolation medium are added to 2 gof harvested cell mass. The resuspended cells and cell aggregates areincubated at 27±2° C. for 4 to 6 hours in the darkness, while shaking itslightly (at 30 to 40 rpm).

Protoplast purification

As soon as the release of at least 1 million protoplasts/ml has takenplace (microscopic inspection), the suspension is sifted through astainless steel or nylon sieve with a mesh size of 200 or 45 μm. Thecombination of a 100 μm and a 60 μm sieve allows for separating the cellaggregates just as well. The protoplast-containing filtrate is examinedmicroscopically. It usually contains 98-99% protoplasts. The rest areundigested single cells. Protoplast preparations with such a degree ofpurity are used for transformation experiments without additionalgradient centrifugation. The protoplasts are sedimented by means ofcentrifugation (100 UpM in the swing-out rotor (100×g, 3 minutes)). Thesupernatant is abandoned and the protoplasts are resuspended in washingsolution 1. The centrifugation is repeated and the protoplasts aresubsequently resuspended in the transformation buffer.

(b) Protoplast transformation

The protoplasts resuspended in the transformation buffer are filled in10 ml portions into 50 ml polyallomer tubes at a titer of 0.5-1×10⁶protoplasts/ml. The DNA used for transformation is dissolved inTris-EDTA (TE) buffer solution. 20 μg plasmid DNA is added to each mlprotoplast suspension. A plasmid which provides for resistance tophosphinotricine is used as vector (cf. e.g. EP 0 513 849). After theaddition of DNA the protoplast suspension is carefully shaken in orderto homogenously distribute the DNA in the solution. Immediatelyafterwards 5 ml PEG solution is added in drops.

By carefully shaking the tubes the PEG solution is distributedhomogenously. Afterwards further 5 ml of PEG solution are added and thehomogenous mixing is repeated. The protoplasts remain in the PEGsolution for 20 minutes at ±2° C. Afterwards the protoplasts aresedimented by centrifuging for 3 minutes (100 g; 1000 Upm). Thesupernatant is abandoned. The protoplasts are washed in 20 ml W5solution by careful shaking and are again subjected to centrifugation.Then they are resuspended in 20 ml protoplast culture medium,centrifuged anew and again resuspended in culture medium. The titer isadjusted to 6-8×10⁵ protoplasts and the protoplasts are cultivated in 3ml portions in Petri dishes (Ø 60 mm, height 15 mm). The Petri dishesare sealed with parafilm and stored in darkness at 25±2°C.

(c) Protoplast culture

During the first 2-3 weeks after the protoplast isolation andtransformation the protoplasts are cultivated without adding freshmedium. As soon as the cells regenerated from the protoplasts havedeveloped into cell aggregates with more than 20 to 50 cells, 1 ml offresh protoplast culture medium, containing sucrose as an osmotic (90g/l), is added.

(d) Selection of transformed maize cells and plant regeneration

3-10 days after adding fresh medium the cell aggregates developed fromthe protoplasts may be plated on Agar media with 100 mg/lL-phosphinothricine. N6-medium with the vitamins of the protoplastculture medium, 90 g/l sucrose and 1.0 mg/l 2,4D is as suitable as ananalogous medium such as a medium with the macro- and micro-nutritivesalts of the MS medium (Murashige and Skoog (1962), see above).

The calli developed from stably transformed protoplasts may grow furtheron the selective medium. After 3 to 5 weeks, preferably 4 weeks thetransgenic calli may be transferred to fresh selection medium which alsocontains 100 mg/l L-phosphinothricine which, however, does no longercontain auxine. Within 3 to 5 weeks approximately 50% of the transgenicmaize calli which had integrated theL-phosphinothricine-acetyl-transferase gene into their genome, start todifferentiate into plants on this medium in the presence ofL-phosphinothricine.

(e) Growing of transgenic regenerative plants

The embryogenical transformed maize tissue is cultivated on hormone-freeN6-medium (Chu C.C. et al., Sci. Sin. 16 (1975), 659) in the presence of5×10⁻⁴ M L-phosphinothricine. On this medium maize embryos, whichexpress the phosphinothricine-acetyl-transferase gene (PAT gene) in asufficiently strong manner, develop into plants. Non-transformed embryosor such with only a very weak PAT activity die down. As soon as theleaves of the in-vitro plants have reached a length of 4 to 6 mm, theymay be transferred into soil. After washing off the Agar residues at theroots the plants are planted into a mixture of clay, sand, vermiculiteand potting soil with the ratio 3:1:1:1 and adapted to the soil cultureat 90-100% of relative atmospheric humidity during the first 3 daysafter planting. The growing is carried out in a climate chamber with a14 hour light period of approximately 25000 lux at the height of theplant at a day/night temperature of 23±1/17±1° C. The adapted plants arecultivated at an 65±5% atmospheric humidity.

4. Radioactive marking of DNA fragments

The radioactive marking of DNA fragments was carried out by means of aDNA-Random Primer Labeling Kits by Boehringer (Germany) according to themanufacturer's instructions.

EXAMPLE 1

Cloning of a cDNA encoding a starch phosphorylase from Zea mays

In order to isolate cDNA molecules encoding a starch phosphorylase frommaize, a cDNA library was constructed within the vector Lambda ZAPII(Stratagene) starting from polyA⁺ RNA from endosperm and packed intophage heads. E.coli cells of the XL1 Blue strain were subsequentlyinfected with the phages containing the cDNA fragments (1×10⁶ pfu) andplated on a medium in Petri dishes with a densitiy of approximately30,000 per 75 cm². After an 8-hour incubation, nitro cellulose membraneswere put on the lysated bacterial culture and removed after one minute.The filters were first incubated in 0.2 M NaOH; 1.5 M NaCl for 2 minutesand then in 0.4 M Tris/HCl pH 7.5 for 2 minutes and finally in 2×SSC for2 minutes. After drying and fixing the DNA by means of UV crosslinking,the filters were incubated in hybridization buffer for 3 hours at 42° C.before a radioactively marked probe was added.

As a probe, use was made of a cDNA from rice encoding a starchphosphorylase from rice (DDBJ accession no. D23280). The hybridizationwas carried out in 2×SSC, 10×Dehnhardt's solution; 50 mM Na₂HPO₄, pH7.2; 0.2% SDS; 5 mM EDTA and 250 μg/ml denaturated herring sperm DNA at48° C.

Hybridizing phage clones were singled out and further purified by meansof standard methods. By means of in vivo excision E.coli clones werederived from positive phage clones. The E.coli clones contained adouble-stranded pBluescript plasmid with the respective cDNA insertions.After examining the size and the restriction pattern of the insertion,plasmid DNA was isolated from suitable clones and subsequentlysequenced, as described in Example 2.

EXAMPLE 2

Sequence analysis of the CDNA insert of the pSTP55 plasmid

The plasmid pSTP55 was isolated from the E.coli clone which was obtainedas described in Example 1, and the sequence of the cDNA insert wasdetermined in a standard routine by means of thedidesoxynucleotide-method (Sanger et al., Proc. Natl. Acad. Sci. USA 74(1977), 5463-5467). The insert has a length of 3320 bp and constitutes apartial cDNA. The nucleotide sequence is indicated under Seq ID No. 1.The corresponding amino acid sequence is indicated under Seq ID No. 2.

A sequence analysis and a comparison with known sequences showed thatthe sequence shown under Seq ID No. 1 is new and encodes a starchphosphorylase from maize. The probably partial coding region exhibitshomology to starch phosphorylases from other organisms, in particular toa starch phosphorylase from rice. Within the framework of thisapplication, the protein encoded by this cDNA insert or by hybridizingsequences is named STP55. By means of this partial cDNA sequence it ispossible for the person skilled in the field of molecular biology toisolate the full-length clones comprising the complete coding region andto determine their sequences without any further ado. In order to do so,e.g. a leaf-specific cDNA expression library from Zea mays, line B73(Stratagene GmbH, Heidelberg) is screened for full-length clonesaccording to standard methods by means of hybridization with a5′-fragment of the cDNA insert of the pSTP55 plasmid (200 bp). Theclones obtained in such are way are subsequently sequenced. On the otherhand the missing terminal 5′-sequences may be obtained by using a5′-Race-method (e.g. of Stratagene or other manufacturers).

Sequence comparisons with cDNA sequences encoding a different plantstarch phosphorylase show that the isolated cDNA encodes a type 2 starchphosphorylase.

EXAMPLE 3

Construction of a vector for plant transformation and generation oftransgenic maize plants

In order to construct a plant transformation vector which encodes theantisense RNA of the nucleic acid molecule of the invention (starchphosphorylase), the vector pUBIbar (see PCT patent applicationWO97/44472) was linearized with the restriction enzyme HpaI anddephosphorylated. The linearized vector was then ligated with a blunted1.7 kb EcoRI/XhoI fragment coding for the starch phosphorylase frommaize, obtained from the pBluescript plasmid in Example 1. In order tocheck the antisense orientation of the ligated cDNA, a restrictionanalysis was performed which results in the expected 600 bp BamHIfragment.

The plant transformation vector (pUBIbar-αpSTP) is shown in FIG. 1.

The vector was then introduced into maize protoplasts by theabove-described method. (100 μg plasmid DNA per 5×10⁷ protoplasts). 350phosphinotricin-resistant clones were obtained. 70 of these wereanalyzed. It was found that 20 separate clones contained the DNAencoding the starch phosphorylase in antisense orientation. All of theseclones were regenerated to transgenic maize plants.

2 3320 base pairs nucleic acid single linear cDNA to mRNA NO NO Zea maysEndosperm pSTP55 CDS 1..2949 1 GGC GAC GAC CAC CTC GCC GCC GCT GCA GCTCGC CAC CGC CTC CCG CCC 48 Gly Asp Asp His Leu Ala Ala Ala Ala Ala ArgHis Arg Leu Pro Pro 1 5 10 15 GCA CGC CTC CTC CTC CGG CGG TGG CGG GGTTCT CCT CCG CGG GCG GTT 96 Ala Arg Leu Leu Leu Arg Arg Trp Arg Gly SerPro Pro Arg Ala Val 20 25 30 CCG GAG GTG GGG TCG CGC CGG GTC GGG GTC GGGGTC GAG GGG CGA TTG 144 Pro Glu Val Gly Ser Arg Arg Val Gly Val Gly ValGlu Gly Arg Leu 35 40 45 CAG CGG CGG GTG TCG GCG CGC AGC GTG GCG AGC GATCGG GAC GTG CAA 192 Gln Arg Arg Val Ser Ala Arg Ser Val Ala Ser Asp ArgAsp Val Gln 50 55 60 GGC CCC GTC TCG CCC GCG GAA GGG CTT CCA AAT GTG CTAAAC TCC ATC 240 Gly Pro Val Ser Pro Ala Glu Gly Leu Pro Asn Val Leu AsnSer Ile 65 70 75 80 GGC TCA TCT GCC ATT GCA TCA AAC ATC AAG CAC CAT GCAGAG TTC GCT 288 Gly Ser Ser Ala Ile Ala Ser Asn Ile Lys His His Ala GluPhe Ala 85 90 95 CCC TTG TTC TCT CCA GAT CAC TTT TCT CCC CTG AAA GCT TACCAT GCG 336 Pro Leu Phe Ser Pro Asp His Phe Ser Pro Leu Lys Ala Tyr HisAla 100 105 110 ACT GCT AAA AGT GTC CTT GAT GCG CTG CTG ATA AAC TGG AATGCG ACA 384 Thr Ala Lys Ser Val Leu Asp Ala Leu Leu Ile Asn Trp Asn AlaThr 115 120 125 TAT GAT TAT TAC AAC AAA ATG AAT GTA AAA CAA GCA TAT TACCTG TCC 432 Tyr Asp Tyr Tyr Asn Lys Met Asn Val Lys Gln Ala Tyr Tyr LeuSer 130 135 140 ATG GAG TTT TTA CAG GGA AGG GCT CTC ACA AAT GCT ATT GGCAAT CTA 480 Met Glu Phe Leu Gln Gly Arg Ala Leu Thr Asn Ala Ile Gly AsnLeu 145 150 155 160 GAG ATT ACT GGT GAA TAT GCA GAA GCA TTA AAA CAA CTTGGA CAA AAC 528 Glu Ile Thr Gly Glu Tyr Ala Glu Ala Leu Lys Gln Leu GlyGln Asn 165 170 175 CTG GAG GAT GTC GCT AGC CAG GAA CCA GAT GCT GCC CTGGGC AAT GGT 576 Leu Glu Asp Val Ala Ser Gln Glu Pro Asp Ala Ala Leu GlyAsn Gly 180 185 190 GGT TTA GGC CGC CTG GCT TCT TGT TTT TTG GAT TCT TTGGCA ACA TTA 624 Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu Asp Ser Leu AlaThr Leu 195 200 205 AAT TAT CCA GCA TTG GGA TAT GGA CTT CGC TAT GAA TATGGC CTC TTT 672 Asn Tyr Pro Ala Leu Gly Tyr Gly Leu Arg Tyr Glu Tyr GlyLeu Phe 210 215 220 AAG CAG ATC ATA ACA AAG GAT GGT CAG GAG GAG ATT GCTGAG AAT TGG 720 Lys Gln Ile Ile Thr Lys Asp Gly Gln Glu Glu Ile Ala GluAsn Trp 225 230 235 240 CTT GAG ATG GGA TAT CCT TGG GAG GTT GTA AGA AATGAT GTC TCT TAT 768 Leu Glu Met Gly Tyr Pro Trp Glu Val Val Arg Asn AspVal Ser Tyr 245 250 255 CCT GTG AAA TTC TAT GGT AAA GTG GTG GAA GGC ACTGAT GGT AGG AAG 816 Pro Val Lys Phe Tyr Gly Lys Val Val Glu Gly Thr AspGly Arg Lys 260 265 270 CAC TGG ATT GGA GGA GAA AAT ATC AAG GCT GTG GCACAT GAT GTC CCT 864 His Trp Ile Gly Gly Glu Asn Ile Lys Ala Val Ala HisAsp Val Pro 275 280 285 ATT CCT GGC TAC AAA ACT AGA ACT ACC AAT AAT CTGCGT CTT TGG TCA 912 Ile Pro Gly Tyr Lys Thr Arg Thr Thr Asn Asn Leu ArgLeu Trp Ser 290 295 300 ACA ACT GTA CCA GCA CAA GAT TTT GAC TTG GCA GCTTTT AAT TCT GGA 960 Thr Thr Val Pro Ala Gln Asp Phe Asp Leu Ala Ala PheAsn Ser Gly 305 310 315 320 GAT CAT ACC AAG GCA TAT GAA GCT CAT CTA AACGCT AAA AAG ATA TGC 1008 Asp His Thr Lys Ala Tyr Glu Ala His Leu Asn AlaLys Lys Ile Cys 325 330 335 CAC ATA TTG TAT CCT GGG GAT GAA TCA CTA GAGGGG AAA GTT CTC CGC 1056 His Ile Leu Tyr Pro Gly Asp Glu Ser Leu Glu GlyLys Val Leu Arg 340 345 350 TTG AAG CAA CAA TAT ACA TTG TGT TCA GCC TCACTA CAG GAC ATC ATT 1104 Leu Lys Gln Gln Tyr Thr Leu Cys Ser Ala Ser LeuGln Asp Ile Ile 355 360 365 GCT CGT TTT GAG AGT AGA GCT GGC GAG TCT CTCAAC TGG GAG GAC TTC 1152 Ala Arg Phe Glu Ser Arg Ala Gly Glu Ser Leu AsnTrp Glu Asp Phe 370 375 380 CCC TCC AAA GTT GCA GTG CAG ATG AAT GAC ACTCAT CCA ACA CTA TGC 1200 Pro Ser Lys Val Ala Val Gln Met Asn Asp Thr HisPro Thr Leu Cys 385 390 395 400 ATT CCT GAG TTA ATG AGA ATA CTG ATG GATGTT AAG GGA TTA AGC TGG 1248 Ile Pro Glu Leu Met Arg Ile Leu Met Asp ValLys Gly Leu Ser Trp 405 410 415 AGT GAG GCA TGG AGT ATT ACA GAA AGA ACCGTG GCA TAC ACT AAC CAT 1296 Ser Glu Ala Trp Ser Ile Thr Glu Arg Thr ValAla Tyr Thr Asn His 420 425 430 ACA GTG CTT CCT GAA GCT CTA GAG AAG TGGAGC TTG GAC ATA ATG CAG 1344 Thr Val Leu Pro Glu Ala Leu Glu Lys Trp SerLeu Asp Ile Met Gln 435 440 445 AAA CTT TTA CCT CGA CAT GTT GAG ATA ATAGAA ACA ATT GAT GAA GAG 1392 Lys Leu Leu Pro Arg His Val Glu Ile Ile GluThr Ile Asp Glu Glu 450 455 460 CTG ATA AAC AAC ATA GTC TCA AAA TAT GGAACC ACA GAT ACT GAA CTG 1440 Leu Ile Asn Asn Ile Val Ser Lys Tyr Gly ThrThr Asp Thr Glu Leu 465 470 475 480 TTG AAA AAG AAG CTG AAA GAG ATG AGAATT CTG GAT AAT GTT GAC CTT 1488 Leu Lys Lys Lys Leu Lys Glu Met Arg IleLeu Asp Asn Val Asp Leu 485 490 495 CCA GCT TCC ATT TCC CAA CTA TTT GTTAAA CCC AAA GAC AAA AAG GAA 1536 Pro Ala Ser Ile Ser Gln Leu Phe Val LysPro Lys Asp Lys Lys Glu 500 505 510 TCT CCT GCT AAA TCA AAG CAA AAG TTACTT GTT AAA TCT TTG GAG ACT 1584 Ser Pro Ala Lys Ser Lys Gln Lys Leu LeuVal Lys Ser Leu Glu Thr 515 520 525 ATT GTT GAG GTT GAG GAG AAA ACT GAGTTG GAA GAG GAG GCG GAG GTT 1632 Ile Val Glu Val Glu Glu Lys Thr Glu LeuGlu Glu Glu Ala Glu Val 530 535 540 CTA TCT GAG ATA GAG GAG GAA AAA CTTGAA TCT GAA GAA GTA GAG GCA 1680 Leu Ser Glu Ile Glu Glu Glu Lys Leu GluSer Glu Glu Val Glu Ala 545 550 555 560 GAA GAA GCG AGT TCT GAG GAT GAGTTA GAT CCA TTT GTA AAG TCT GAT 1728 Glu Glu Ala Ser Ser Glu Asp Glu LeuAsp Pro Phe Val Lys Ser Asp 565 570 575 CCT AAG TTA CCA AGA GTT GTC CGAATG GCA AAC CTC TGT GTT GTT GGT 1776 Pro Lys Leu Pro Arg Val Val Arg MetAla Asn Leu Cys Val Val Gly 580 585 590 GGG CAT TCA GTA AAT GGT GTA GCTGAA ATT CAC AGT GAA ATT GTG AAA 1824 Gly His Ser Val Asn Gly Val Ala GluIle His Ser Glu Ile Val Lys 595 600 605 CAG GAT GTG TTC AAC AGC TTC TATGAG ATG TGG CCA ACT AAA TTT CAG 1872 Gln Asp Val Phe Asn Ser Phe Tyr GluMet Trp Pro Thr Lys Phe Gln 610 615 620 AAT AAA ACA AAT GGA GTG ACT CCCAGG CGT TGG ATC CGG TTT TGT AAT 1920 Asn Lys Thr Asn Gly Val Thr Pro ArgArg Trp Ile Arg Phe Cys Asn 625 630 635 640 CCT GCA TTA AGT GCA TTA ATTTCA AAG TGG ATT GGT TCT GAT GAC TGG 1968 Pro Ala Leu Ser Ala Leu Ile SerLys Trp Ile Gly Ser Asp Asp Trp 645 650 655 GTG CTT AAT ACA GAC AAA CTGGCA GAA CTG AAG AAG TTT GCT GAT AAT 2016 Val Leu Asn Thr Asp Lys Leu AlaGlu Leu Lys Lys Phe Ala Asp Asn 660 665 670 GAA GAT CTG CAT TCA GAG TGGCGT GCT GCT AAG AAG GCT AAC AAA ATG 2064 Glu Asp Leu His Ser Glu Trp ArgAla Ala Lys Lys Ala Asn Lys Met 675 680 685 AAG GTT ATT TCT CTT ATA AGGGAG AAG ACA GGA TAT ATT GTC AGT CCA 2112 Lys Val Ile Ser Leu Ile Arg GluLys Thr Gly Tyr Ile Val Ser Pro 690 695 700 GAT GCA ATG TTT GAT GTG CAGGTG AAA AGG ATA CAT GAA TAT AAG CGG 2160 Asp Ala Met Phe Asp Val Gln ValLys Arg Ile His Glu Tyr Lys Arg 705 710 715 720 CAG CTG CTA AAT ATC CTTGGA ATT GTC TAC CGC TAC AAG AAG ATG AAA 2208 Gln Leu Leu Asn Ile Leu GlyIle Val Tyr Arg Tyr Lys Lys Met Lys 725 730 735 GAA ATG AGC ACA GAA GAAAGA GCA AAG AGC TTT GTT CCA AGG GTA TGC 2256 Glu Met Ser Thr Glu Glu ArgAla Lys Ser Phe Val Pro Arg Val Cys 740 745 750 ATA TTC GGT GGG AAA GCATTT GCC ACA TAT ATA CAG GCA AAA AGG ATC 2304 Ile Phe Gly Gly Lys Ala PheAla Thr Tyr Ile Gln Ala Lys Arg Ile 755 760 765 GTT AAA TTT ATT ACA GATGTG GCA GCT ACC GTG AAC CAT GAT TCA GAC 2352 Val Lys Phe Ile Thr Asp ValAla Ala Thr Val Asn His Asp Ser Asp 770 775 780 ATT GGA GAT TTG TTG AAGGTC GTA TTT GTT CCA GAC TAT AAT GTT AGT 2400 Ile Gly Asp Leu Leu Lys ValVal Phe Val Pro Asp Tyr Asn Val Ser 785 790 795 800 GTT GCC GAG GCA CTAATT CCT GCC AGT GAA TTG TCA CAG CAT ATC AGT 2448 Val Ala Glu Ala Leu IlePro Ala Ser Glu Leu Ser Gln His Ile Ser 805 810 815 ACT GCT GGA ATG GAAGCT AGT GGG ACC AGT AAC ATG AAG TTT GCA ATG 2496 Thr Ala Gly Met Glu AlaSer Gly Thr Ser Asn Met Lys Phe Ala Met 820 825 830 AAC GGT TGC ATT CTTATT GGA ACT TTA GAT GGT GCA AAT GTG GAG ATC 2544 Asn Gly Cys Ile Leu IleGly Thr Leu Asp Gly Ala Asn Val Glu Ile 835 840 845 AGA GAG GAG GTT GGAGAA GAA AAC TTT TTC CTT TTT GGT GCA GAG GCA 2592 Arg Glu Glu Val Gly GluGlu Asn Phe Phe Leu Phe Gly Ala Glu Ala 850 855 860 CAT GAA ATT GCT GGTTTG CGG AAA GAA AGA GCC GAG GGA AAG TTT GTG 2640 His Glu Ile Ala Gly LeuArg Lys Glu Arg Ala Glu Gly Lys Phe Val 865 870 875 880 CCT GAC CCA AGATTT GAG GAG GTT AAG GAA TTT GTC CGC AGT GGT GTC 2688 Pro Asp Pro Arg PheGlu Glu Val Lys Glu Phe Val Arg Ser Gly Val 885 890 895 TTT GGG ACT TACAGC TAT GAT GAA TTG ATG GGG TCT TTG GAA GGA AAT 2736 Phe Gly Thr Tyr SerTyr Asp Glu Leu Met Gly Ser Leu Glu Gly Asn 900 905 910 GAA GGT TAC GGACGT GCA GAT TAT TTC CTT GTT GGC AAG GAC TTC CCC 2784 Glu Gly Tyr Gly ArgAla Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro 915 920 925 AGC TAT ATT GAATGC CAA GAA AAA GTT GAT GAG GCG TAC CGA GAT CAG 2832 Ser Tyr Ile Glu CysGln Glu Lys Val Asp Glu Ala Tyr Arg Asp Gln 930 935 940 AAG TTA TGG ACAAGG ATG TCT ATC CTC AAC ACG GCT GGC TCA TCC AAG 2880 Lys Leu Trp Thr ArgMet Ser Ile Leu Asn Thr Ala Gly Ser Ser Lys 945 950 955 960 TTC AGC AGCGAT AGG ACG ATT CAT GAG TAC GCC AAG GAT ATC TGG GAT 2928 Phe Ser Ser AspArg Thr Ile His Glu Tyr Ala Lys Asp Ile Trp Asp 965 970 975 ATC AGC CCTGCC ATC CTT CCC TAGACCAGGT GGATATCAGG TTCTTTCGCC 2979 Ile Ser Pro AlaIle Leu Pro 980 TATATTTCTG TGAACCCTCA GGATCAAGGA ACAGTTGGTG ACGACATTAATTTGCCTCAG 3039 CCCCTTAGCA GGAAGCGCTG GTCACCTCAG TTTTGTGTAG ACAAAATCTAGGCATCGATA 3099 AATGATGGGA CTATGCATGG TATTTTGGCA GCACTGTTCA GTACCTTGCCTTTTAAATCT 3159 GGTTTTTGGT GTGTGTGTGT GTAAGCTAAT AAATGTCGAG GCAGGATTGTAGGAACACCA 3219 TTGATCATTT GGCTCGCTGG TGAACCTGGT GACGTATGGT GTAATTAGTAGTTGTTTGCC 3279 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A 3320 983amino acids amino acid linear protein 2 Gly Asp Asp His Leu Ala Ala AlaAla Ala Arg His Arg Leu Pro Pro 1 5 10 15 Ala Arg Leu Leu Leu Arg ArgTrp Arg Gly Ser Pro Pro Arg Ala Val 20 25 30 Pro Glu Val Gly Ser Arg ArgVal Gly Val Gly Val Glu Gly Arg Leu 35 40 45 Gln Arg Arg Val Ser Ala ArgSer Val Ala Ser Asp Arg Asp Val Gln 50 55 60 Gly Pro Val Ser Pro Ala GluGly Leu Pro Asn Val Leu Asn Ser Ile 65 70 75 80 Gly Ser Ser Ala Ile AlaSer Asn Ile Lys His His Ala Glu Phe Ala 85 90 95 Pro Leu Phe Ser Pro AspHis Phe Ser Pro Leu Lys Ala Tyr His Ala 100 105 110 Thr Ala Lys Ser ValLeu Asp Ala Leu Leu Ile Asn Trp Asn Ala Thr 115 120 125 Tyr Asp Tyr TyrAsn Lys Met Asn Val Lys Gln Ala Tyr Tyr Leu Ser 130 135 140 Met Glu PheLeu Gln Gly Arg Ala Leu Thr Asn Ala Ile Gly Asn Leu 145 150 155 160 GluIle Thr Gly Glu Tyr Ala Glu Ala Leu Lys Gln Leu Gly Gln Asn 165 170 175Leu Glu Asp Val Ala Ser Gln Glu Pro Asp Ala Ala Leu Gly Asn Gly 180 185190 Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu Asp Ser Leu Ala Thr Leu 195200 205 Asn Tyr Pro Ala Leu Gly Tyr Gly Leu Arg Tyr Glu Tyr Gly Leu Phe210 215 220 Lys Gln Ile Ile Thr Lys Asp Gly Gln Glu Glu Ile Ala Glu AsnTrp 225 230 235 240 Leu Glu Met Gly Tyr Pro Trp Glu Val Val Arg Asn AspVal Ser Tyr 245 250 255 Pro Val Lys Phe Tyr Gly Lys Val Val Glu Gly ThrAsp Gly Arg Lys 260 265 270 His Trp Ile Gly Gly Glu Asn Ile Lys Ala ValAla His Asp Val Pro 275 280 285 Ile Pro Gly Tyr Lys Thr Arg Thr Thr AsnAsn Leu Arg Leu Trp Ser 290 295 300 Thr Thr Val Pro Ala Gln Asp Phe AspLeu Ala Ala Phe Asn Ser Gly 305 310 315 320 Asp His Thr Lys Ala Tyr GluAla His Leu Asn Ala Lys Lys Ile Cys 325 330 335 His Ile Leu Tyr Pro GlyAsp Glu Ser Leu Glu Gly Lys Val Leu Arg 340 345 350 Leu Lys Gln Gln TyrThr Leu Cys Ser Ala Ser Leu Gln Asp Ile Ile 355 360 365 Ala Arg Phe GluSer Arg Ala Gly Glu Ser Leu Asn Trp Glu Asp Phe 370 375 380 Pro Ser LysVal Ala Val Gln Met Asn Asp Thr His Pro Thr Leu Cys 385 390 395 400 IlePro Glu Leu Met Arg Ile Leu Met Asp Val Lys Gly Leu Ser Trp 405 410 415Ser Glu Ala Trp Ser Ile Thr Glu Arg Thr Val Ala Tyr Thr Asn His 420 425430 Thr Val Leu Pro Glu Ala Leu Glu Lys Trp Ser Leu Asp Ile Met Gln 435440 445 Lys Leu Leu Pro Arg His Val Glu Ile Ile Glu Thr Ile Asp Glu Glu450 455 460 Leu Ile Asn Asn Ile Val Ser Lys Tyr Gly Thr Thr Asp Thr GluLeu 465 470 475 480 Leu Lys Lys Lys Leu Lys Glu Met Arg Ile Leu Asp AsnVal Asp Leu 485 490 495 Pro Ala Ser Ile Ser Gln Leu Phe Val Lys Pro LysAsp Lys Lys Glu 500 505 510 Ser Pro Ala Lys Ser Lys Gln Lys Leu Leu ValLys Ser Leu Glu Thr 515 520 525 Ile Val Glu Val Glu Glu Lys Thr Glu LeuGlu Glu Glu Ala Glu Val 530 535 540 Leu Ser Glu Ile Glu Glu Glu Lys LeuGlu Ser Glu Glu Val Glu Ala 545 550 555 560 Glu Glu Ala Ser Ser Glu AspGlu Leu Asp Pro Phe Val Lys Ser Asp 565 570 575 Pro Lys Leu Pro Arg ValVal Arg Met Ala Asn Leu Cys Val Val Gly 580 585 590 Gly His Ser Val AsnGly Val Ala Glu Ile His Ser Glu Ile Val Lys 595 600 605 Gln Asp Val PheAsn Ser Phe Tyr Glu Met Trp Pro Thr Lys Phe Gln 610 615 620 Asn Lys ThrAsn Gly Val Thr Pro Arg Arg Trp Ile Arg Phe Cys Asn 625 630 635 640 ProAla Leu Ser Ala Leu Ile Ser Lys Trp Ile Gly Ser Asp Asp Trp 645 650 655Val Leu Asn Thr Asp Lys Leu Ala Glu Leu Lys Lys Phe Ala Asp Asn 660 665670 Glu Asp Leu His Ser Glu Trp Arg Ala Ala Lys Lys Ala Asn Lys Met 675680 685 Lys Val Ile Ser Leu Ile Arg Glu Lys Thr Gly Tyr Ile Val Ser Pro690 695 700 Asp Ala Met Phe Asp Val Gln Val Lys Arg Ile His Glu Tyr LysArg 705 710 715 720 Gln Leu Leu Asn Ile Leu Gly Ile Val Tyr Arg Tyr LysLys Met Lys 725 730 735 Glu Met Ser Thr Glu Glu Arg Ala Lys Ser Phe ValPro Arg Val Cys 740 745 750 Ile Phe Gly Gly Lys Ala Phe Ala Thr Tyr IleGln Ala Lys Arg Ile 755 760 765 Val Lys Phe Ile Thr Asp Val Ala Ala ThrVal Asn His Asp Ser Asp 770 775 780 Ile Gly Asp Leu Leu Lys Val Val PheVal Pro Asp Tyr Asn Val Ser 785 790 795 800 Val Ala Glu Ala Leu Ile ProAla Ser Glu Leu Ser Gln His Ile Ser 805 810 815 Thr Ala Gly Met Glu AlaSer Gly Thr Ser Asn Met Lys Phe Ala Met 820 825 830 Asn Gly Cys Ile LeuIle Gly Thr Leu Asp Gly Ala Asn Val Glu Ile 835 840 845 Arg Glu Glu ValGly Glu Glu Asn Phe Phe Leu Phe Gly Ala Glu Ala 850 855 860 His Glu IleAla Gly Leu Arg Lys Glu Arg Ala Glu Gly Lys Phe Val 865 870 875 880 ProAsp Pro Arg Phe Glu Glu Val Lys Glu Phe Val Arg Ser Gly Val 885 890 895Phe Gly Thr Tyr Ser Tyr Asp Glu Leu Met Gly Ser Leu Glu Gly Asn 900 905910 Glu Gly Tyr Gly Arg Ala Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro 915920 925 Ser Tyr Ile Glu Cys Gln Glu Lys Val Asp Glu Ala Tyr Arg Asp Gln930 935 940 Lys Leu Trp Thr Arg Met Ser Ile Leu Asn Thr Ala Gly Ser SerLys 945 950 955 960 Phe Ser Ser Asp Arg Thr Ile His Glu Tyr Ala Lys AspIle Trp Asp 965 970 975 Ile Ser Pro Ala Ile Leu Pro 980

What is claimed is:
 1. An isolated nucleic acid molecule encoding aprotein with the biological activity of a starch phosphorylase frommaize wherein the nucleic acid molecule comprises a nucleic acidsequence selected from the group consisting of (a) a nucleic acidsequence encoding a protein comprising the amino acid sequence of SEQ IDNO: 2; (b) a nucleic acid sequence of SEQ ID NO: 1 or a complementarynucleic acid sequence thereof; (c) a nucleic acid sequence which hasmore than 80% overall sequence identity to the coding region of SEQ IDNO: 1; and (d) a nucleic acid sequence, the nucleotide sequence of whichdeviates from the sequence of the nucleic acid sequence according to (b)or (c) due to the degeneracy of the genetic code.
 2. The nucleic acidmolecule according to claim 1 which is a DNA molecule.
 3. The DNAmolecule according to claim 2 which is a cDNA molecule.
 4. The nucleicacid molecule according to claim 1 which is an RNA molecule.
 5. A vectorcomprising the nucleic acid molecule according to any one of claims 1 to3.
 6. The vector according to claim 5, wherein the nucleic acid moleculeis linked in sense-orientation to regulatory elements that ensure thetranscription and synthesis of a translatable RNA in prokaryotic oreukaryotic cells.
 7. A transgenic host cell comprising the nucleic acidmolecule according to any one of claims 1 to 4 or comprising a vectorcomprising the nucleic acid molecule, wherein the nucleic acid moleculein the vector is optionally linked in sense-orientation to regulatoryelements that ensure the transcription and synthesis of a translatableRNA in prokaryotic or eukaryotic cells, or a cell derived from thetransgenic host cell, wherein said derived cell comprises the nucleicacid molecule according to any one of claims 1 to 4 or comprises avector comprising the nucleic acid molecule.
 8. A method for theproduction of a protein with the biological activity of a starchphosphorylase from maize, comprising the steps of cultivating the hostcell according to claim 7 under conditions that allow for the synthesisof the protein and isolating the protein from the cultivated cellsand/or the culture medium.
 9. A transgenic plant cell comprising thenucleic acid molecule according to any one of claims 1 to 4 orcomprising a vector comprising the nucleic acid molecule, wherein thenucleic acid molecule comprised by the vector is optionally linked insense-orientation to regulatory elements that ensure the transcriptionand synthesis of a translatable RNA in prokaryotic or eukaryotic cells,or a cell which is derived from such a transgenic cell, wherein saidderived cell comprises the nucleic acid molecule according to any one ofclaims 1 to 4 or comprises a vector comprising the nucleic acid moleculewherein the nucleic acid molecule encoding the protein with thebiological activity of a starch phosphorylase is under the control ofregulatory elements allowing for the transcription of a translatableMRNA in plant cells.
 10. A plant comprising the transgenic plant cellaccording to claim
 9. 11. The plant according to claim 10 which is astarch-storing plant.
 12. The plant according to claim 11 which is amaize plant.
 13. Propagation material of a plant comprising the plantcell according to claim
 9. 14. A method for the production of a modifiedstarch comprising the step of extracting the starch from the plantaccording to claim 10 and/or from starch-storing parts of the plant. 15.A transgenic maize plant cell, wherein the activity of a starchphosphorylase from maize is reduced, wherein the reduction is achievedby sense expression of a nucleic acid molecule that has been introducedinto the cell, wherein the nucleic acid molecule comprises a nucleicacid sequence selected from the group consisting of: (a) a nucleic acidsequence encoding a protein comprising the amino acid sequence of SEQ IDNO: 2; (b) a nucleic acid sequence of SEQ ID NO: 1 or a complementarynucleic acid sequence thereof; (c) a nucleic acid sequence that has morethan 80% overall sequence identity to the coding region of SEQ ID NO: 1or a complementary nucleic acid sequence thereof; and (d) a nucleic acidsequence, the nucleotide sequence of which deviates from the sequence ofthe nucleic acid sequence according to (b) or (c) due to the degeneracyof the genetic code.
 16. A transgenic maize plant cell, wherein theactivity of a starch phosphorylase from maize is reduced, wherein thereduction is achieved by sense expression of a nucleic acid moleculethat has been introduced into the cell, wherein the nucleic acidmolecule comprises a part of a nucleic acid sequence selected from thegroup consisting of: (a) a nucleic acid sequence encoding a proteincomprising the amino acid sequence of SEQ ID NO:2; (b) a nucleic acidsequence of SEQ ID NO: 1 or a complementary nucleic acid sequencethereof; (c) a nucleic acid sequence that has more than 80% overallsequence identity to the coding region of SEQ ID NO: 1 or acomplementary nucleic acid sequence thereof; and (d) a nucleic acidsequence, the nucleotide sequence of which deviates from the sequence ofthe nucleic acid sequence according to (b) or (c) due to the degeneracyof the genetic code; wherein said part is of a length sufficient toreduce the activity of said starch phosphorylase.
 17. The transgenicmaize plant cell according to claim 15, wherein the nucleic acidmolecule comprises a nucleic acid sequence that has more than 90%overall sequence identity to the coding region of SEQ ID NO: 1 or acomplementary strand thereof.
 18. The transgenic maize plant cellaccording to claim 17, wherein the nucleic acid molecule comprises thenucleic acid sequence of SEQ ID NO:
 1. 19. The transgenic maize plantcell according to claim 16, wherein the nucleic acid molecule comprisesa nucleic acid sequence that has more than 90% overall sequence identityto the coding region of SEQ ID NO: 1 or a complementary strand thereof.20. The transgenic maize plant cell according to claim 19, wherein thenucleic acid molecule comprises the nucleic acid sequence of SEQ IDNO:
 1. 21. A maize plant comprising the transgenic maize plant cellaccording to any of claims 15-20, wherein the activity of said proteinis reduced.
 22. Propagation material of the maize plant of claim
 21. 23.A method for production of a modified starch comprising the step ofextracting the starch from the plant acording to claim 17 and/or fromstarch-storing parts of the plant.
 24. Propagation material according toclaim 13, wherein the plant is selected from the group consisting of astarch-synthesizing plant, a starch-storing plant and a maize plant. 25.The method according to claim 14, wherein the plant is selected from thegroup consisting of a starch-synthesizing plant, a starch-storing plantand a maize plant.
 26. A maize plant comprising the plant cell accordingto any one of claims 15-20.
 27. Propagation material of a maize plantcomprising the cell according to any one of claims 15-20.
 28. A methodfor the production of a modified starch comprising the step ofextracting the starch from the plant according to claim 26 and/or fromstarch-storing parts of the plant.
 29. The plant according to claim 11,wherein the plant is selected from the group consisting of rye, barley,oats, wheat, rice, maize, pea, cassava and potato.
 30. The methodaccording to claim 25, wherein the starch-storing plant is selected fromthe group consisting of rye, barley, oats, wheat, rice, maize, pea,cassava and potato.
 31. The isolated nucleic acid molecule according toclaim 1, wherein the nucleic acid sequence has more than 90% sequenceidentity to the entire coding region of SEQ ID NO:
 1. 32. The isolatednucleic acid molecule according to claim 1, wherein the nucleic acidsequence is the coding region of SEQ ID NO:
 1. 33. An isolated nucleicacid molecule comprising the nucleic acid sequence of SEQ ID NO:
 1. 34.The isolated nucleic acid molecule according to claim 1, wherein thenucleic acid sequence encodes a protein comprising the amino acidsequence of SEQ ID NO:
 2. 35. The isolated nucleic acid moleculeaccording to claim 1, wherein the nucleic acid sequence deviates fromthe nucleic acid sequence of a nucleic acid molecule that has more than90% overall sequence identity to the entire coding region of SEQ ID NO:1 due to the degeneracy of the genetic code.
 36. The isolated nucleicacid molecule according to claim 1, wherein the nucleic acid sequencedeviates from the nucleic acid sequence of a nucleic acid molecule thatis the entire coding region of SEQ ID NO: 1 due to the degeneracy of thegenetic code.